Infection, Genetics and Evolution 11 (2011) 44–51
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Infection, Genetics and Evolution
journal homepage: www.elsevier.com/locate/meegid
Identification and lineage genotyping of South American trypanosomes using
fluorescent fragment length barcoding
P.B. Hamilton a,*, M.D. Lewis b, C. Cruickshank a, M.W. Gaunt b, M. Yeo b, M.S. Llewellyn b,
S.A. Valente c, F. Maia da Silva d, J.R. Stevens a, M.A. Miles b, M.M.G. Teixeira d
a
Biosciences, College of Life and Environmental Sciences, University of Exeter, Prince of Wales Road, Exeter EX4 4PS, United Kingdom
Department of Pathogen Molecular Biology Unit, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom
Instituto Evandro Chagas, Belém, PA 67030-070, Brazil
d
Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP 05508-900, Brazil
b
c
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 20 August 2010
Received in revised form 14 October 2010
Accepted 15 October 2010
Available online 26 October 2010
Trypanosoma cruzi and Trypanosoma rangeli are human-infective blood parasites, largely restricted to
Central and South America. They also infect a wide range of wild and domestic mammals and are
transmitted by a numerous species of triatomine bugs. There are significant overlaps in the host and
geographical ranges of both species. The two species consist of a number of distinct phylogenetic
lineages. A range of PCR-based techniques have been developed to differentiate between these species
and to assign their isolates into lineages. However, the existence of at least six and five lineages within T.
cruzi and T. rangeli, respectively, makes identification of the full range of isolates difficult and time
consuming. Here we have applied fluorescent fragment length barcoding (FFLB) to the problem of
identifying and genotyping T. cruzi, T. rangeli and other South American trypanosomes. This technique
discriminates species on the basis of length polymorphism of regions of the rDNA locus. FFLB was able to
differentiate many trypanosome species known from South American mammals: T. cruzi cruzi, T. cruzi
marinkellei, T. dionisii-like, T. evansi, T. lewisi, T. rangeli, T. theileri and T. vivax. Furthermore, all five T.
rangeli lineages and many T. cruzi lineages could be identified, except the hybrid lineages TcV and TcVI
that could not be distinguished from lineages III and II respectively. This method also allowed
identification of mixed infections of T. cruzi and T. rangeli lineages in naturally infected triatomine bugs.
The ability of FFLB to genotype multiple lineages of T. cruzi and T. rangeli together with other
trypanosome species, using the same primer sets is an advantage over other currently available
techniques. Overall, these results demonstrate that FFLB is a useful method for species diagnosis,
genotyping and understanding the epidemiology of American trypanosomes.
ß 2010 Elsevier B.V. All rights reserved.
Keywords:
Co-infection
Genetic diversity
Vector
Chagas disease
Protozoa
Kinetoplastid
1. Introduction
Trypanosoma cruzi and Trypanosoma rangeli are the two species
of human-infective trypanosomes occurring in overlapping areas
of South and Central America. T. cruzi causes Chagas disease, a
condition that affects at least 8 million people, with 100 million at
risk and 14,000 deaths annually (Jannin and Salvatella, 2006).
Despite recent advances in disrupting vector transmission in
Southern Cone countries, this disease remains a major public
health problem in Latin America (Schofield et al., 2006; Miles et al.,
2009). In regions endemic for Chagas disease, T. cruzi circulates
between humans and domestic animals and is transmitted by
domiciliated triatomine bugs. However, infection by T. cruzi is a
* Corresponding author. Tel.: +44 01392 263917; fax: +44 01392 263700.
E-mail address: [email protected] (P.B. Hamilton).
1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.meegid.2010.10.012
widespread zoonosis, ranging from the southern half of the USA to
the southernmost countries of South America (Marcili et al.,
2009c). T. rangeli is not believed to cause disease in humans. A high
prevalence of T. rangeli in humans has been reported in Central
America and northwestern South America, where concomitant
infections and serological cross-reactivity with T. cruzi make
diagnosis of Chagas disease difficult (Vallejo et al., 2009). Both T.
cruzi and T. rangeli have a wide mammalian host range and are
transmitted by a large diversity of triatomine bugs, although only
species of the genus Rhodnius transmits T. rangeli (Maia da Silva
et al., 2007; Vallejo et al., 2009).
Molecular studies have revealed high genetic diversity in T.
cruzi and T. rangeli, with isolates of both species distributed into
several lineages, also called discrete taxonomic units (DTU) within
T. cruzi (Stevens et al., 1999; Maia da Silva et al., 2007; Miles et al.,
2009; Vallejo et al., 2009). At least six lineages of T. cruzi cruzi have
been described using molecular markers including RAPDs, SSU
P.B. Hamilton et al. / Infection, Genetics and Evolution 11 (2011) 44–51
rRNA gene sequences, microsatellites and mitochondrial genes
(e.g. Brisse et al., 2001; Machado and Ayala, 2001; Freitas et al.,
2005; Westenberger et al., 2005; Miles et al., 2009). These lineages
differ in their host range, ecotope and geographical distribution
(Miles et al., 2009) and are potentially associated with variable
forms of Chagas disease (Anez et al., 2004). T. cruzi lineages until
recently designed as TcI and TcIIa–e (Brisse et al., 2001; Miles et al.,
2009) were recently redesigned as follows: TcI, TcII (former TcIIb),
TcIII (TcIIc), TcIV (TcIIa), TcV (TcIId) and TcVI (TcIIe) (Zingales et al.,
2009).
Accurate identification of T. cruzi and T. rangeli, and their
respective lineages is important for diagnosis and understanding
their epidemiology. In addition, other species of mammalian
trypanosome can be found in the vertebrate hosts of these species,
and there are potentially species that are yet to be discovered
(Stevens et al., 1999; Maia da Silva et al., 2008; Marcili et al.,
2009a,c; Cavazzana et al., 2010). Distinguishing between T. cruzi
and T. rangeli lineages is still problematic, especially in regions
where man, wild reservoirs and triatomines can be found infected
with different combinations of isolates from different lineages of
both T. cruzi and T. rangeli (Yeo et al., 2005, 2007; Vallejo et al.,
2009). Morphology is insufficient for species identification,
particularly in mixed infections in vectors. In mixed cultures T.
cruzi prevails over T. rangeli and, after successive passages,
typically only one lineage of T. cruzi is selected (Yeo et al., 2007;
Maia da Silva et al., 2008). PCR assays have increased sensitivity
and accuracy of diagnosis of T. cruzi, allowing identification
directly from tissue samples, and triatomine guts and faeces
(Hamano et al., 2001; Virreira et al., 2003). Several PCR-based
methods are able to differentiate T. cruzi from T. rangeli, including
PCR with species-specific primers developed from several genomic
regions: kDNA minicircles (Avila et al., 1991); telomeric repeats
(Chiurillo et al., 2003); repetitive DNA (Vargas et al., 2000);
randomly amplified polymorphic DNA (RAPD)-derived markers
(Maia da Silva et al., 2004a), spliced leader gene (Maia da Silva
et al., 2007) and Cathepsin L-like gene (Ortiz et al., 2009). Length
differences in a region of the 24S rRNA gene permitted the
identification of the three common trypanosomatid species in
triatomines: T. cruzi, T. rangeli and Blastocrithidia triatoma (Schijman et al., 2006).
The most widely used methods for differentiating between T.
cruzi lineages are based on polymorphism of 24S alpha rDNA and
spliced leader DNA, although these methods are unable to
distinguish all lineages (Souto et al., 1996; Fernandes et al.,
2001). Indeed, some studies have shown that use of a single
molecular marker can lead to misclassification of T. cruzi isolates
(Brisse et al., 2001; Burgos et al., 2007; Marcili et al., 2009a,b,c). The
five lineages of T. rangeli can be distinguished through length and
sequence polymorphisms of the internal transcribed spacer (ITS)
rDNA regions, cathepsin L-like and spliced leader (SL) genes (Maia
da Silva et al., 2004b, 2007, 2009; Ortiz et al., 2009).
Fluorescent fragment length barcoding (FFLB) is a method that
discriminates species by size polymorphisms in specific regions of
the 18S and 28S ribosomal RNA genes (Hamilton et al., 2008). It has
been applied to the identification of African trypanosomes, both in
tsetse flies and in vertebrates, and its use has led to the discovery of
new strains and species (Adams et al., 2008, 2009, 2010; Adams
and Hamilton, 2008; Hamilton et al., 2009). It has proved to be
quick, accurate, and able to detect mixed infections of up to three
different strains (Hamilton et al., 2008; Adams et al., 2009). The
high diversity and complexity of T. cruzi and T. rangeli suggest that
many genotypes remain to be described, especially from the
generally poorly investigated sylvatic vertebrate and invertebrate
hosts of unexplored geographical regions and ecotopes. Here we
apply this technique to the issue of species and lineage
identification of the American trypanosomes, T. cruzi and T. rangeli.
45
Our results provide evidence that FFLB is a useful tool for
elucidating the genetic diversity present within these species
and for better understanding of the epidemiology of American
trypanosomes.
2. Materials and methods
2.1. T. cruzi and T. rangeli isolates
The isolates of T. cruzi and T. rangeli were selected for this study
to represent the broad genetic diversity found in a range of
vertebrate and vector species from their full geographical range
(Table 2). They represented the six recognised lineages of T. cruzi
cruzi, one new genotype of this species associated with bats (TCbat)
and T. c. marinkellei, the subspecies most closely related to T. cruzi
cruzi (Stevens et al., 1999) and thought to be restricted to bats from
Central and South America (Marcili et al., 2009a; Cavazzana et al.,
2010). Isolates of the five currently recognised lineages of T. rangeli
(Maia da Silva et al., 2004b, 2007, 2009; Ortiz et al., 2009) were also
selected for this study. Identity of species/isolates was as
confirmed in previous studies (Maia da Silva et al., 2008; Marcili
et al., 2009a,b,c).
2.2. Fluorescent fragment length barcoding
FFLB analysis was carried out using primers and the PCR
programme described previously (Hamilton et al., 2008), except
REDTaq1 DNA Polymerase (Sigma) was used. All DNA samples
were isolated from cultured trypanosomes. A total of four primer
sets were used (two sets within the 18S rRNA gene and two within
the 28S a rRNA gene) to create a barcode for each sample,
consisting of the lengths of the four amplified regions. These were
then compared to barcodes from other trypanosomes obtained in
previous studies (Hamilton et al., 2008, 2009; Adams et al., 2009).
3. Results and discussion
3.1. Identification of American trypanosomes using fluorescent
fragment length barcoding
In this study, we evaluated the use of fluorescent fragment
length barcoding (FFLB) for identification and diagnosis of species
of American trypanosomes and genotyping of lineages of T. cruzi
and T. rangeli. We compared isolates from all recognised major
lineages of T. cruzi and T. rangeli. All isolates examined gave peaks
with the four primer sets. Figs. 1 and 2 show example FFLB profiles
from a range of species. The method was able to differentiate T.
cruzi from T. rangeli independent of lineages of these two species,
as the size ranges of 18S1, 18S3 and 28S1 did not overlap between
the two species (Tables 1 and 2). The FFLB patterns of T. cruzi and T.
rangeli also differed from those of several other species that are
known from South American mammals: T. evansi, T. dionisii-like, T.
lewisi, T. theileri and T. vivax (Table 1). Additionally, all T. rangeli
lineages and the several of T. cruzi lineages could be distinguished,
demonstrating that FFLB could be useful for epidemiological
studies. Indeed, two loci 18S1 and 28S, used together, were
sufficient to discriminate all lineages except the two T. cruzi hybrid
lineages, while the other loci often provided additional confidence
in the results.
3.2. Identification of T. cruzi lineages
Sixty-four T. cruzi cruzi isolates belonging to the six established
T. cruzi lineages (TcI–TcVI), together with two genotypes that are
apparently restricted to bats: TCbat and T. c. marinkellei, were
characterised using the FFLB method (Tables 1 and 2 and Fig. 1).
[()TD$FIG]
46
P.B. Hamilton et al. / Infection, Genetics and Evolution 11 (2011) 44–51
Table 1
Fluorescent fragment length barcoding profiles of American trypanosomes.
Fragment lengths (bp)
18S1
18S3
28S1
242–246
334–349
T. cruzi TcI (formerly I)
294–309a
T. cruzi TcII (formerly IIb)
302–303
240–241
348–351
T. cruzi TcIII (formerly IIc)
300–301
237
332–336
T. cruzi TcIV (formerly IIa)
286–288
239–240
321–325
T. cruzi TcV (formerly IId)
300
236–237
334
T. cruzi TcVI (formerly IIe)
302–303
240–241
350
T. cruzi TcBAT
317
239
335
All T. cruzi cruzi
286–317
236–246
321–351
T. cruzi marinkellei
280
236
311
T. rangeli TrA
267–269
223
297–298
T. rangeli TrB
261
224
298
T. rangeli TrC
266
223
302
T. rangeli TrD
268
223
305
T. rangeli TrE
267
223
300
All T. rangeli
261–269
223–224
297–305
Other trypanosomes/trypanosomatids found in South America
T. dionisii-like
259–261
227
304–310
T. theileric
233–234
216
250–251
209
234
291–292
T. evansid
T. lewisi
239
216
266
e
T. vivax
189
199
–f
Unidentified
234
212
257
trypanosomatid
344
222
Blastocrithidia triatomag
28S2
197–199
213–215
188–189
204–207
189–190
214
189
188–215
196
193–194
188
191
191, 193b
186
186–194
203–208
183
199
195
172
194
a
294–309 either indicates multiple peaks within this range for individual
isolates or, different isolates of the strain give different sized peaks within the range.
b
191, 193 indicates two peaks for one isolate.
c
From Hamilton et al. (2009).
d
From Hamilton et al. (2008).
e
From Adams et al. (2009).
f
Dash indicates that no peak was detected consistently for multiple isolates.
g
predicted from DNA sequence AF153037; no sequences for the 28S rDNA are
currently available for this species.
Fig. 1. Example electropherograms from T. cruzi. x axis, size of fragment in base
pairs; y axis, fluorescence intensity. Small peaks without numbers are size standard.
Tcm = T. cruzi marinkellei.
TcI was clearly distinguished from all other lineages using one
locus, 28S2. However, there was significant heterogeneity within
this lineage. All four loci varied in size, with the largest size range
(15 bp) at 18S1. Most TcI isolates, including three that were cloned
(Xe5740, SJM39, M7) gave up to three peaks at two loci, 18S1 and
28S1 (Fig. 1). This could result from the presence of multiple
divergent ribosomal copies within the genome of some TcI isolates,
although the multiple peaks may result from PCR artifacts. Recent
studies have shown high variability and distinct genotypes within
TcI on the basis of the polymorphism of the intergenic region of
spliced leader gene (Cura et al., in press) and DNA microsatellites
(Llewellyn et al., 2009b). However, while in this study isolates
originated from throughout the range of TcI, no obvious
geographical pattern of FFLB profiles was apparent. TcI is the
most common lineage in sylvatic cycles from North (southern
USA), Central and South America, transmitted mainly by Rhodnius
species. This lineage predominates as an agent of human infection
from the Amazon basin northwards, where it is the main cause of
Chagas disease in endemic areas of Venezuela, Colombia, Panama
and Mexico (Miles et al., 1981; Bosseno et al., 2006; Burgos et al.,
2007; Samudio et al., 2007; Anez et al., 2009; Mejia-Jaramillo et al.,
2009). In Brazil, this lineage is reported to infect humans in rural
endemic areas (Teixeira et al., 2006) and in the Amazonia region
(Miles et al., 1981; Marcili et al., 2009c; Valente et al., 2009).
TcII (formerly IIb) is common in domestic transmission cycles in
Southern Cone countries of South America and is mainly
transmitted by T. infestans. TcII could not be clearly distinguished
from TcVI using FFLB, although at the loci 28S1 and 28S2 some
fragment sizes were found only in TcII.
TcIII (IIc) has a widespread distribution, occurring from
Venezuela and Brazilian Amazonia to southern Brazil, Argentina,
and Paraguay, transmitted by Triatoma and Panstrongylus species
mainly in sylvatic and peridomestic cycles (Yeo et al., 2005; Freitas
et al., 2006; Llewellyn et al., 2009a; Marcili et al., 2009b; Miles
et al., 2009). TcIII showed heterogeneous profiles, and could not be
distinguished from TcV, although some 28S2 fragment sizes were
restricted to TcIII. The 28S1 loci of three Brazilian isolates, JA2cl2,
M6241cl6 and TCC1437 were longer (336 bp, compared to 332–
334 bp) than the other isolates typed, although the significance of
this is not known (Table 2).
TcIV (IIa) is common in wild monkeys and Rhodnius in the
Brazilian Amazonia, where it has been sporadically reported from
human cases of oral infection (Miles et al., 1981; Maia da Silva
et al., 2008; Marcili et al., 2009c). This lineage showed heterogeneous profiles: the North American isolate (92122102r) gave a
distinct profile from the eight South American isolates of TcIV at
two loci: 18S1 (288, compared to 286 for South American isolates)
and 28S2 (207, compared to 204), corroborating that they are
closely related, yet distinct (Barnabe et al., 2001; Marcili et al.,
2009a; Bosseno et al., 2009).
The hybrid lineages TcV (IId) and TcVI (IIe) occur in Bolivia,
Paraguay, Chile, Argentina and southern Brazil, and predominate in
humans, domestic and synanthropic (animals that live in close
association with humans) mammals and triatomines (Brisse et al.,
P.B. Hamilton et al. / Infection, Genetics and Evolution 11 (2011) 44–51
47
Table 2
Origins of trypanosomes used in the study and their FFLB profiles.
Species/
subspecies
Type/
lineage
Strain/
isolate
Location
Host/vector
T.
T.
T.
T.
T.
T.
T.
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
TcI
TcI
TcI
TcI
TcI
TcI
TcI
(I)
(I)
(I)
(I)
(I)
(I)
(I)
Bajo Calima, Colombia
Cojedes, Venezuela
Dtto Federal, Venezuela
Georgia, USA
Para, Brazil
Cotopachi, Bolivia
Florida, USA
Kinkajou Potus flavus
Human Homo sapiens
Human Homo sapiens
Opossum Didelphis marsupialis
Opossum Philander opossum
Grass mouse Akodon boliviensis
Triatomine Triatoma sanguisuga
308
303
305
309
299
308
309
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
TcI
TcI
TcI
TcI
TcI
TcI
TcI
TcI
TcI
TcI
TcI
TcI
TcI
TcI
(I)
(I)
(I)
(I)
(I)
(I)
(I)
(I)
(I)
(I)
(I)
(I)
(I)
(I)
458
11006
11124
93070103P
b2026
COTMA22
FLORID
AC1D12
JR cl4
M16
M7b
PALDA21
SJM34b
SJM39b
SJM41b
TEV55
TCC1358c
TCC1360
TCC1367
TCC1380
TCC1397
14844TCC1591
TCC1010
15818TCC1612
15907TCC1613
TCC651
TCC758
Anzoátegui, Venezuela
Barinas, Venezuela
Barinas, Venezuela
Chaco, Argentina
Beni, Bolivia
Beni, Bolivia
Beni, Bolivia
Chaco, Argentina
Amazonas, Brazil
Para, Brazil
Para, Brazil
Para, Brazil
Para, Brazil
Para, Brazil
Human Homo sapiens
Opossum Didelphis marsupialis
Opossum Didelphis marsupialis
Opossum Didelphis albiventris
Opossum Didelphis marsupialis
Opossum Didelphis marsupialis
Opossum Philander opossum
Triatomine Triatoma infestans
Triatomine Rhodnius brethesi
Triatomine Rhodnius pictipes
Triatomine Rhodnius robustus
Triatomine Rhodnius robustus
Triatomine Rhodnius pictipes
Human Homo sapiens
Rondonia, Brazil
Para, Brazil
T. cruzi cruzi
T. cruzi cruzi
TcI (I)
TcI (I)
T. cruzi cruzi
TcI (I)
T. cruzi cruzi
T. cruzi cruzid
TcI (I)
TcI (I)
T. cruzi cruzi
TcI (I)
T. cruzi cruzie
FFLB profile
18S1
18S3
28S1
28S2
244
245
245
245
244
244
245
343
342
342
339
341
345
339
197
199
199
198
198
197
198
306 (302, 308)
306
306
307
307
308 (302, 304)
305
302
294, 298
294, 298, 301
294 (298)
295 (301)
294, 298
295, 297
242, 243
242 (244)
244
244
244
244
244
244
244
244
244
244
243
244
198
199
199
198
197
197
198
198
198
198
198
198
198
198
Opossum Didelphis marsupialis
Human Homo sapiens
302, 309
297, 294
244
243
336, 344
334, 339
339 (334)
349
343
343 (345)
345
345
(335) 340
335
334
334
335, 340
335, 337,
342
342, 345
335
Para, Brazil
Human Homo sapiens
294, 298
243
339
198
Rondonia, Brazil
Amazonas, Brazil
Triatomine Rhodnius robustus
Triatomine Rhodnius brethesi
244
239, 244
342
321, 335,
341
342
198
198, 204
194, 198
198
198
(306)a
(309)
(301)
(305)
198
198
Louisiana, USA
Opossum Didelphis marsupialis
TcI (I)
USAO
POSSUM
Xe1313
307
286, 302,
304, 309
303 (309)
Para, Brazil
Opossum Philander opossum
234, 302
245246
212, 244
T. cruzi cruzi
T. cruzi cruzi
TcI (I)
TcI (I)
Xe5167
Xe5740b
Para, Brazil
Para, Brazil
Opossum Didelphis marsupialis
Opossum Didelphis marsupialis
294 (299)
294 (301)
244
244
257,
335
344
335
T. cruzi cruzi
TcII (IIb)
302
240
349
215
TcII (IIb)
TcII (IIb)
TcII (IIb)
Presidente Hayes,
Paraguay
Bahia, Brazil
Cuncumen, Chile
San Martin,
Boqueron, Paraguay
Triatomine Triatoma infestans
T. cruzi cruzi
T. cruzi cruzi
T. cruzi cruzi
Chaco23
cl4
Esm cl3
IVV cl4
Pot7a cl1
Human Homo sapiens
Human Homo sapiens
Triatomine Triatoma infestans
302
302
303
241
240
240
348
351
349
213
214
214
T.
T.
T.
T.
T.
TcIII
TcIII
TcIII
TcIII
TcIII
JA2 cl2
M6241 cl6
SABP19 cl1
SJMO18
SMA8
Opossum Monodelphis sp.
Human Homo sapiens
Triatomine Triatoma infestans
Armadillo Dasypus novemcinctus
Armadillo Dasypus novemcinctus
301
301
301
300
300
237
237
237
237
237
336
336
334
333
333
189
189
189
189
189
Armadillo Dasypus novemcinctus
300
237
333
189
Rodent Proechimys iheringi
300
237
332333
336
333
188-189
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
(IIc)
(IIc)
(IIc)
(IIc)
(IIc)
T. cruzi cruzi
TcIII (IIc)
SMA9
T. cruzi cruzi
TcIII (IIc)
TCC135
Amazonas, Brazil
Para, Brazil
Vitor, Peru
Beni, Bolivia
Santa Maria de
Apere, Bolivia
Santa Maria de
Apere, Bolivia
Sao Paulo, Brazil
T. cruzi cruzi
T. cruzi cruzi
TcIII (IIc)
TcIII (IIc)
TCC1437
TCC712
Para, Brazil
Amazonas, Brazil
Rodent Proechimys longicaudatus
Marsupial Monodelphis brevicaudata
301
301
237
237
T. cruzi cruzi
T. cruzi cruzi
T. cruzi cruzi
TcIV (IIa)
TcIV (IIa)
TcIV (IIa)
Georgia, USA
Venezuela
Amapa, Brazil
Raccoon Procyon lotor
Squirrel monkey Saimiri sciureus
Human Homo sapiens
288
286
286
239
239
239
T. cruzi cruzi
TcIV (IIa)
Para, Brazil
Human Homo sapiens
286
T.
T.
T.
T.
T.
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
TcIV
TcIV
TcIV
TcIV
TcIV
(IIa)
(IIa)
(IIa)
(IIa)
(IIa)
92122102r
Saimiri3 cl1
4766TCC1434
3475TCC1441
TCC338
TCC668
TCC759
TCC760
X10610 cl5
Acre, Brazil
Rondonia, Brazil
Amazonas, Brazil
Amazonas, Brazil
Guárico, Venezuela
Monkey Saguinus labiatus
Triatomine Rhodnius robustus
Triatomine Rhodnius brethesi
Triatomine Rhodnius brethesi
Human Homo sapiens
T.
T.
T.
T.
T.
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
cruzi
TcV
TcV
TcV
TcV
TcV
(IId)
(IId)
(IId)
(IId)
(IId)
92-80 cl2
Para6 cl4
Bertha
TCC656
NR cl3
Santa Cruz, Bolivia
Paraguari, Paraguay
Santa Cruz, Bolivia
Santa Cruz, Bolivia
Chile
Human Homo sapiens
Triatomine Triatoma infestans
Human Homo sapiens
Human Homo sapiens
Human Homo sapiens
199
189
189
207
204
204
239
322
321
323,
325
322
286
286
286
286
286
239
239
239
239
240
321
321
321
321
321
204
204
204
204
204
300
300
300
300
300
237
237
236
237
237
334
334
334
334
334
189
189
190
189
189
204
P.B. Hamilton et al. / Infection, Genetics and Evolution 11 (2011) 44–51
48
Table 2 (Continued )
Species/
subspecies
Type/
lineage
Strain/
isolate
Location
Host/vector
FFLB profile
18S1
18S3
28S1
28S2
T. cruzi cruzi
TcV (IId)
Vinch101
cl1
Limari, Chile
Triatomine Triatoma infestans
300
237
334
189
T. cruzi cruzi
TcVI (IIe)
Chaco9 cl15
Triatomine Triatoma infestans
303
241
350
214
T. cruzi cruzi
TcVI (IIe)
CL Brener
Triatomine Triatoma infestans
302
240
350
214
T. cruzi cruzi
T. cruzi cruzi
TcVI (IIe)
TcVI (IIe)
P251 cl7
Tula cl2
Presidente Hayes,
Paraguay
Rio Grande do Sul,
Brazil
Cochabamba, Bolivia
Tulahuen, Chile
Human Homo sapiens
Human Homo sapiens
302
302
240
240
350
350
214
214
T. cruzi cruzi
T. cruzi cruzi
Tcbat
Tcbat
TCC793
TCC1122
Sao Paulo, Brazil
Sao Paulo, Brazil
Bat Myotis levis
Bat Myotis albescens
317
317
239
239
335
335
189
189
T cruzi marinkellei
T cruzi marinkellei
TCC344
TCC501
Rondonia, Brazil
Rondonia, Brazil
Bat Carollia perspicillata
Bat Carollia perspicillata
280
280
236
236
311
311
196
196
T. dionisii-like
T. dionisii-like
TCC211
TCC495
Sao Paulo, Brazil
Sao Paulo, Brazil
Bat Eptesicus brasiliensis
Bat Carolia perspicillata
261
259
227
227
307, 310
304
203
208
T.
T.
T.
T.
rangeli
rangeli
rangeli
rangeli
TrA
TrA
TrA
TrA
San Augustin
TCC220
TCC369
TCC701
Colombia
Para, Brazil
Rondonia, Brazil
Rondonia, Brazil
Human Homo sapiens
Monkey Saimiri sciureus
Opssum Didelphis marsupialis
Triatomine Rhodnius robustus
267
269
269
269
223
223
223
223
298
297
298
297
194
194
193
193
T.
T.
T.
T.
rangeli
rangeli
rangeli
rangeli
TrB
TrB
TrB
TrB
TCC010
AM80
TCC207
TCC236
Para, Brazil
Amazonas, Brazil
Acre, Brazil
Acre, Brazil
Anteater Tamandua tetradactyla
Human Homo sapiens
Monkey Cebuella pygmaea
Monkey Saguinus f. weddelli
261
261
261
261
224
224
224
224
298
298
298
298
188
188
188
188
T.
T.
T.
T.
rangelif
rangelif
rangeli
rangeli
TrC
TrC
TrC
TrC
TCC1250
TCC1252
TCC1254
PG
Panama
Panama
Panama
Panama
Triatomine Rhodnius pallescens
Triatomine Rhodnius pallescens
Triatomine Rhodnius pallescens
Human Homo sapiens
266, 301
266, 301
266
266
223, 244
223, 244
223
223
302, 341
302, 341
302
302
191, 198
191, 198
191
191
T. rangeli
TrD
SC58
Santa Catarina, Brazil
Rodent Echimys dasithrix
268
223
305
191, 193
T. rangeli
TrE
TCC643
Bat Platyrrhinus lineatus
267
223
300
186
T. rangeli
T. rangeli
TrE
TrE
TCC1182
TCC1224
Mato Grosso do
Sul, Brazil
Amazonas, Brazil
Amazonas, Brazil
Triatomine Rhodnius pictipes
Triatomine Rhodnius pictipes
267
267
223
223
300
300
186
186
a
b
c
d
e
f
Values in parentheses are <50% the height of ‘main peak’ from the same locus.
Cloned isolate.
TCC = Trypanosomatid Culture Collection.
Mixed infection with TCIV.
Mixed infection with unidentified trypanosome.
Mixed infection with TCI.
2003; Yeo et al., 2005; Corrales et al., 2009). The hybrid isolates of
TcVI and TcV could be distinguished by three loci but shared patterns
with TcII and TcIII respectively. This result is perhaps not surprising
because these lineages are products of hybridization of TcII and TcIII
(Freitas et al., 2006; Miles et al., 2009). There is geographic overlap in
the distribution of these hybrid lineages and their parental lineages,
especially for TcII and TcV/VI (domestic cycles across the Southern
Cone). The overlap is more limited for TcIII, as it is rare in domestic
cycles where TcV and TcVI are found, but there are reports of TcIII
being sympatric with TcV and TcVI in Paraguay and Argentina
(Chapman et al., 1984; Cardinal et al., 2008). Therefore further
primers, such as those targeting ITS rDNA (Marcili et al., 2009a), or
PCR-RFLP assays (Lewis et al., 2009), would be necessary to
discriminate these hybrids from the parent lineages. Nevertheless,
as the traditional genotyping method (Souto et al., 2006) shows
combined TcII/III profiles for the hybrid lineages, FFLB can be used to
differentiate mixed infections from hybrids.
The newly discovered T. cruzi genotype that is so far apparently
restricted to bats, TCbat, showed unique FFLB pattern, in
agreement with its placement in a separated cluster in phylogenetic studies (Marcili et al., 2009a). Two other South American bat
trypanosomes, T. c marinkellei and T. dionisii-like, also gave unique
patterns (Table 1). The two isolates of T. dionisii-like differed in
their profiles (Table 2); there is considerable heterogeneity of T.
dionisii-like in South America and these two isolates belong to
distinct genotypes of this species (Cavazzana et al., 2010).
3.3. Identification of Trypanosoma rangeli lineages
Seventeen T. rangeli isolates belonging to the five lineages (TrA–
TrE) were genotyped using FFLB (Tables 1 and 2 and Fig. 2). These
lineages were previously established by phylogenetic analysis
using ITS rDNA, spliced leader and CatL-like gene sequences (Maia
da Silva et al., 2004b, 2007, 2009; Ortiz et al., 2009). All lineages
could be differentiated, with each lineage giving a distinct
combination of fragment sizes at the four loci (Table 1). The FFLB
patterns of TrB were most divergent, and the sizes of three of the
four loci differed from the other T. rangeli lineages, in agreement
with phylogenetic studies (Maia da Silva et al., 2007, 2008; Ortiz
et al., 2009). The geographical distributions of T. rangeli lineages are
related to the ecogeographical structure of the Rhodnius vector
species, with lineage divergence associated with sympatric vectors
(Maia da Silva et al., 2004b, 2007, 2009; Vallejo et al., 2009): TrA
circulates from Brazil to Guatemala and is related to both domestic
and sylvatic cycles of species of the R. prolixus complex, and is
commonly found infecting man; TrB so far includes only sylvatic
isolates from humans and wild mammals from Brazilian Amazonia
and is associated with the R. brethesi complex. TrC is related to
[()TD$FIG]
P.B. Hamilton et al. / Infection, Genetics and Evolution 11 (2011) 44–51
49
expected from mixed infections revealed that many of the common
mixed infections that occur in natural conditions such as TcI with
TcIII could be identified. T. rangeli, lineages TrA and TrC (Panama,
Costa Rica and northwest Colombia), and TrA and TrB (Amazonian
region) can infect the same vertebrate hosts but not the same
vectors, however, all these lineages could be distinguished using
FFLB. In studies of African trypanosomes, the application of FFLB
has resulted in discovery of novel species and genotypes (Hamilton
et al., 2008, 2009; Adams et al., 2009). In the current study, a
barcode was obtained from a recent TcI culture from a marsupial
(Fig. 2), Philander opossum, which differed from those of other
trypanosome species examined in this study, and may represent a
previously undescribed trypanosome species.
4. Conclusions
Fig. 2. Example electropherograms from T. rangeli, T. dionisii-like and mixed
infections. See legend to Fig. 1 for further information.
domestic and sylvatic cycles of the R. pallescens complex
circulating in humans, and domestic and wild mammals in
Panama, Costa Rica and Colombia. Lineage TrD is known from
rodents from southern Brazil; a presumed Rhodnius sp. vector of
this lineage is unknown. TrE has so far only been found in bats and
R. pictipes from Central and Amazon regions in Brazil.
In conclusion, FFLB is a useful tool for the identification of a
wide range of American trypanosomes and for lineage identification of T. cruzi and T. rangeli. The technique is quick and sensitive, as
it relies on amplification of relatively small regions of DNA and
fluorescence detection and is able to differentiate mixed infections.
In previous studies, FFLB genotypes have been obtained from DNA
isolated directly from blood (Adams et al., 2009) and digestive
tracts/proboscides of insects (Hamilton et al., 2008). In the present
study, mixed cultures with two species or two lineages were
detected using DNA from primary cultures from the guts of
triatomines, so it seems likely that American trypanosomes could
also be identified without prior use of culturing, thus avoiding
selection of species/genotypes. The present study also highlighted
the limitations of the FFLB technique, particularly for characterisation of T. cruzi strains. These were length polymorphism within
single isolates, hybrid strains and their evolutionary predecessors
giving matching profiles and the inability to identify TcI
sublineages. However, additional regions, such as the intergenic
spacer of spliced-leader genes, which is known to vary in length
between some TcI sublineages (Cura et al., in press), could also be
included to provide further discrimination. FFLB also requires
access to a DNA sequencer and knowledge of the system, so is not
suitable for diagnosis in rural settings. Nevertheless, its ability
differentiate many known (and potentially unknown) species and
several of their lineages, using the same primer sets, is unique and
offers an advantage over other established methods for identifying
American trypanosomes and should facilitate large scale diagnostics and epidemiological studies.
Acknowledgements
3.4. Identification of Blastocrithidia triatomae
Many triatomines, particularly Triatoma spp., may also carry
Blastocrithidia triatomae, a trypanosomatid parasite of triatomines
that apparently does not have a vertebrate host. As no DNA from
this species was available, the FFLB profile was estimated from the
18S rDNA sequence (AF153037). The 18S1 locus (344 bp) is 27 bp
longer than all trypanosome species examined in this study, so this
would not be mistaken for T. cruzi or T. rangeli.
3.5. Mixed infections and novel genotypes
Mixed infections of different strains of T. rangeli and T. cruzi are
common in both mammalian and triatomine hosts. In previous
studies, mixed infections of African trypanosomes have been
readily detected using FFLB (Hamilton et al., 2008; Adams et al.,
2009). In this study, FFLB detected previously unidentified mixed
infections in primary cultures from gut contents of triatomine
bugs: TcI and TrC in two R. pallescens (Table 2 and Fig. 2); and TcI
and TcIV in R. brethesi (Table 2). Examination of FFLB patterns
We thank M. Tibayrenc, C. Barnabe, P. Diosque, Hernan
Carrasco, Angela C.V. Junqueira, Vera C. Valente, Arlei Marcili,
Luciana Lima and for samples used in this study. Funding from
Wellcome Trust, CNPq-Brazil and EC FP7 project ChagasEpiNet. We
would also like to thank two anonymous reviewers for their helpful
comments.
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